Fig 1.

Vascular endothelial growth factor (VEGF) autocrine loop is enhanced upon spheroid growth of synovial sarcoma (SS). (a) Immunohistochemical analysis of SS clinical specimen with VEGF-A. Nuclei were counterstained with hematoxylin. Scale bar = 100 μm. (b) Quantity of VEGF-A secreted by Yamato-SS under 2-D or spheroid (3-D) culture conditions, as determined by ELISA. Culture medium was collected every 24 h for 4 days (left panel, n = 3; *P < 0.005). Quantification of VEGFA mRNA of Yamato-SS by quantiative RT-PCR under 2-D or spheroid culture conditions from day 1 to day 7 (right panel, n = 3; *P < 0.05). A.U., arbitrary unit (the same l applies hereafter). (c) Immunoblot analysis of hypoxia-inducible factor-1α (HIF-1α) in Yamato-SS cells cultured under 2-D or spheroid culture conditions for 3 days. (d) Immunoblot analysis of VEGF receptor 2 (VEGFR2) in Yamato-SS cells cultured under 2-D or spheroid culture conditions, from day 1 to day 7. (e) Yamato-SS cells were cultured under 2-D conditions with or without recombinant human VEGF-A (rhVEGF-A; 10 ng/mL) or spheroid conditions for 5 days, followed by immunoprecipitation (IP) with anti-VEGFR2 and blotting for p-tyrosine (p-Tyr) and VEGFR2. (f) Immunofluorescent staining of Yamato-SS spheroids with proliferation marker proliferating cell nuclear antigen (PCNA; top panel), VEGF-A (middle panel), and VEGFR2 (bottom panel). Nuclei were counterstained with Hoechst33342 (blue). Scale bar = 100 μm. (g) Dose-dependence response to bevacizumab (Bev) treatment for 2 weeks, as seen with a soft agar assay. Colonies >200 μm are shown (n = 3; *P < 0.05, **P < 0.005). (h) Rescue experiments, using exogenously added rhVEGF-A (10 ng/mL), in Yamato-SS cells treated with Bev. Colonies >200 μm are shown (n = 3; *P < 0.005, **P < 0.05). M.W., molecular weight.